Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

<p>Abstract</p> <p>Background</p> <p>For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in <it>Rhipicephalus appendiculatus </it>and <it>Rhipicephalus (Boophilus) microplus </it>ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against <it>R. microplus </it>were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against <it>R. appendiculatus</it>. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between <it>R. microplus </it>and <it>R. appendiculatus</it>. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility.</p> <p>Results</p> <p>The transcription levels of nine potential reference genes: β-actin (ACTB), β-tubulin (BTUB), elongation factor 1α (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of <it>R. microplus </it>and <it>R. appendiculatus</it>. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in <it>R. appendiculatus </it>and <it>R. microplus </it>revealed a more continuous Bm86 antigen abundance in <it>R. microplus </it>throughout its one-host life cycle compared to the three-host tick <it>R. appendiculatus </it>where large variations were observed between different life stages.</p> <p>Conclusion</p> <p>Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole <it>R. microplus </it>and <it>R. appendiculatus </it>ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in <it>R. appendiculatus</it>.</p

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37±38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31±32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3±6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas.The Science and Technology Directorate of the United States Department of Homeland Security (DHS) under Award Instrument Number: D15PC0027.http://www.plosone.orgam2017Veterinary Tropical Disease

Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations

<p>Abstract</p> <p>Background</p> <p>The cattle ticks, <it>Boophilus </it>spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant <it>B. microplus </it>Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for <it>B. annulatus </it>Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>Recombinant Ba86 (Israel strain) was used to immunize cattle to test its efficacy for the control of <it>B. annulatus </it>(Mercedes, Texas, USA strain) and <it>B. microplus </it>(Susceptible, Mexico strain) infestations. Bm86 (Gavac and Mozambique strain) and adjuvant/saline were used as positive and negative controls, respectively. Vaccination with Ba86 reduced tick infestations (71% and 40%), weight (8% and 15%), oviposition (22% and 5%) and egg fertility (25% and 50%) for <it>B. annulatus </it>and <it>B. microplus</it>, respectively. The efficacy of both Ba86 and Bm86 was higher for <it>B. annulatus </it>than for <it>B. microplus</it>. The efficacy of Ba86 was higher for <it>B. annulatus </it>(83.0%) than for <it>B. microplus </it>(71.5%). The efficacy of Bm86 (Gavac; 85.2%) but not Bm86 (Mozambique strain; 70.4%) was higher than that of Ba86 (71.5%) on <it>B. microplus</it>. However, the efficacy of Bm86 (both Gavac and Mozambique strain; 99.6%) was higher than that of Ba86 (83.0%) on <it>B. annulatus</it>.</p> <p>Conclusion</p> <p>These experiments showed the efficacy of recombinant Ba86 for the control of <it>B. annulatus </it>and <it>B. microplus </it>infestations in cattle and suggested that physiological differences between <it>B. microplus </it>and <it>B. annulatus </it>and those encoded in the sequence of Bm86 orthologs may be responsible for the differences in susceptibility of these tick species to Bm86 vaccines.</p

De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick’s enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum